DNA methylation is an essential regulator of transcription, chromatin structure, and development in mammalian cells mediated by the action of DNA methyltransferases DNMT1, DNMT3A, and DNMT3B. While critical for normal homeostasis, deregulated methylation patterns, characterized by repetitive element and gene body hypomethylation and promoter region hypermethylation, are a hallmark of tumor cells and an early event in tumorigenesis. Recent exciting findings, including whole genome mapping of DNMT binding sites from our laboratory and the marked enrichment of methylation in exons, pinpoint important functions for DNA methylation and DNMTs in intragenic regions. Identification of the new ICF syndrome gene ZBTB24 points to a novel class of DNMT targeting proteins. Collectively these and other findings make this an ideal moment to renew our long-time focus on de novo methyltransferase DNMT3B and take our research examining how it is targeted throughout the genome into a new and potentially paradigm-shifting direction. The central hypothesis to be tested in this application is that de novo methyltransferase DNMT3B is a major regulator of genomic DNA methylation patterns in normal cells and that disruption of its functions through aberrant targeting contributes to DNA methylation defects in cancer. Specifically in this application, we propose that DNMT3B targeting is regulated by aspects of chromatin and sequence-specific features/factors (including ZBTB24), and that a major function of these interactions in normal cells is to recruit DNA methylation to intragenic loci to regulate alternative RNA splicing. We will test this hypothesis with three specific aims. In aim 1 we will define chromatin and DNA sequence determinants characteristic of loci bound by DNMT3B and targeted for DNA methylation in a model differentiation system. In aim 2 we will investigate how DNMT3B is recruited to intragenic regions and how its DNA methylation activity influences alternative splicing (specifically exon skipping). Finally, in aim 3 we will characterize the newly discovered ICF Syndrome gene ZBTB24 (ZNF450) and determine how it regulates DNMT3B and DNA methylation targeting. Addressing this hypothesis has the potential to radically change how we think of the functions of methylation (regulating splicing) and how methylation is targeted throughout the genome (via ZBTB family members) under normal and pathological conditions. Intragenic loci are an evolutionarily conserved seat for DNA methylation. Our studies will shed new light on how the frequent, but poorly studied, hypomethylation that occurs in cancer cells may drive aberrant cell growth through altered RNA splicing rather than through transcriptional silencing. This is expected to positively affect human health by allowing for a more complete understanding of the functions of methylation in different regions of the genome, which should enhance our ability to develop novel therapies to correct aberrant methylation or use it as a drug target.